This is a competitive renewal of NIH grant DK083567. The overall objective of this grant is focused on the novel role of Rho-kinase (ROCK) in the regulation of glucose metabolism and insulin signaling. During the past funding period, we identified ROCK1 as a key regulator of insulin sensitivity and adiposity, providing new insights into the pathogenesis of type 2 diabetes and obesity. In particular, we demonstrated that peripheral ROCK1 controls glucose metabolism and insulin sensitivity, whereas hypothalamic ROCK1 dominantly regulates feeding behavior and body-weight homeostasis. In the next cycle, we will determine the physiological role of hepatic ROCK1 in glucose and lipid metabolism, insulin sensitivity, and energy expenditure, with the long-term goal of finding new therapeutic targets for type 2 diabetes and obesity. Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes and is often found in obesity-associated metabolic diseases. Our preliminary data suggest the importance of ROCK1 action in hepatic metabolism that is critical for the development of insulin resistance and non-alcoholic fatty acid liver diseases. We found that selective deletion of ROCK1 in the liver prevents obesity, insulin resistance, and hepatic steatosis in diet-induced obese mice. These effects are most likely due to decreased hepatic de novo lipogenesis and increased whole-body energy expenditure independent of food intake. Interestingly, hepatic and circulating FGF21 levels are greatly increased by hepatic ROCK1 deficiency in which hepatic autophagic signaling remains defective. Furthermore, we found that ROCK1 physically binds to LXR and increases LXR serine phosphorylation, suggesting the involvement of ROCK1 in LXR action. We thus hypothesize that hepatic ROCK1 activation is necessary for the metabolic regulation of glucose and lipid homeostasis that is linked to autophagy-mediated FGF21 action and LXR activation. Aim 1 will determine the functional importance of hepatic ROCK isoforms in regulating whole- body glucose and lipid homeostasis. Aim 2 will elucidate the mechanisms by which hepatic ROCK1 regulates energy expenditure. Aim 3 will establish the cellular mechanisms for the effects of ROCK1 on hepatic lipid accumulation. To accomplish these aims, we will employ state-of-the-art biochemical, molecular, cellular, and metabolic physiological techniques, including genetically engineering tissue-specific transgenic mouse models and adenovirus-mediated gene transfer systems. These studies will provide a unique opportunity to establish a new mechanism in which ROCK1 is a key determinant of hepatic metabolism and may offer further insights into the pathogenesis of obesity-linked metabolic diseases, which may lead to a novel therapeutic target for the treatment of obesity and diabetes.